ABSTRACT ? PROJECT 2 About 90% of all HIV transmissions occur via mucosal exposures; HIV first encounters mucosal fluids, in which IgAs are important components. Although more IgA is produced daily than all other immunoglobulins combined, its role in preventing HIV acquisition is unclear. Humans have both IgA1 and IgA2. Using passive intrarectal (i.r.) immunization in rhesus monkeys (RMs) against i.r. challenge with a clade C simian-human immunodeficiency virus (SHIV-C), we tested monoclonal dimeric IgA1 (dIgA1) and dIgA2 targeting the same HIV Env epitope. Surprisingly, dIgA1 protected significantly better than dIgA2, although both mAbs neutralized the tier 1 SHIV-C challenge virus equally well in vitro. Better virion capture in vitro was linked to better protection in vivo. This led to the following hypotheses: 1) dIgA1 can trap more virions in the mucosal lumen than dIgA2; and 2) non-neutralizing dIgAs with strong virion binding will prevent mucosal SHIV-C transmission. Next, we combined a low intravenous (i.v.) dose of the same mAb in the IgG1 form with a suboptimal dIgA2 dose (given i.r.) and obtained 100% protection. In stark contrast, i.v. administration of the IgG1 mAb alone at the same dose yielded 0%, and i.r. administration of the dIgA2 mAb alone gave 17% protection, respectively. The remarkable synergy (0%+17%=100%) implied that active vaccination should induce mucosal dIgA as a first line of defense against HIV as well as systemic IgG. Lastly, cell-mediated immunity (CMI) can provide a third line of defense after virus has invaded submucosal tissues. Such a defensive strategy is best described by the military term ?defense-in-depth? ? an approach to defend a vital core by well-armed, multiple lines of defense each of which can serve as backup in case the frontline is breached. The overall goal of Project 2 is to test the key hypothesis that defense-in-depth will protect against mucosal tier 2 SHIV-C transmission ? even if individual defense modalities are insufficient by themselves. The Specific Aims are to: 1. Compare passive i.r. immunization with monoclonal dIgAs that neutralize the tier 2 SHIV-C challenge virus with monoclonal dIgAs that only bind virions without neutralization. 2. Combine two monoclonal dIgAs targeting different Env epitopes in a passive i.r. immunization regimen. 3. Test whether adding i.v. passive immunization with an IgG1 mAb to an i.r. administered dIgA mAb will improve protection against i.r. tier 2 R5 SHIV-C challenge. 4. Test all layers of defense by combining treatment with i.r. dIgA mAb, i.v. IgG1 mAb, and active lymph node- targeted vaccination to induce mucosal CMI. RMs will be given (in order of time of administration): a) amphiphile-vaccination to induce anti-Gag and anti-Tat T-cell responses (see Project 3); b) i.v. IgG1 mAb with effector function and an epitope specificity that is inducible by current immunogens, and c) the i.r. monoclonal dIgA or dIgA combination that yielded optimal protection in earlier experiments.